Base information on PCR, Phases of PCR and Application

1. Polymerase Chain Reaction
(PCR)
Dr. T. Ramesh
Assistant Professor of Zoology
Vivekananda College

2. Contents
Introduction
Principle
Techniques of PCR
Application

3. Introduction
PCR, polymerase chain reaction, is an in-vitro technique-
Large quantities of specified DNA
Cell free Amplification techniques-Synthesis of multiple
copies of DNA
1966, Thomas Brock discovers Thermus aquaticus
1984, Kary Mullis postulated the concept of PCR (Nobel
Prize in 1993)
1985, Saiki publishes the first application of PCR
1985, Cetus Corp. Scientists isolate Thermostable Taq
Polymerase (from T. aquaticus), which revolutionized
PCR

4. PCR- Principles
1. Denaturation
2. Renaturation
3. Synthesis
 Double strand DNA- denatured
 Each strand then allow to
Hybridize with primers
 Template duplex is used for
synthesis of DNA
 Repeat these again and again
with optimum condition of
Temp.-Multiple copies can
produced
PCR instrument

5. Reaction Components
DNA template- The double stranded DNA of interest separated
from the sample.
Primers- Short pieces of single stranded DNA (often 20-30 bp)
which are complementary to the 3’ ends.
Enzyme- Usually a thermostable Taq polymerase that does not
rapidly denature at high temperatures.
Deoxynucleotides- Single units of the bases A, T, G, and C
(dATP, dTTP, dGTP, dCTP).
Buffers- Magnesium & Potassium

6. dsDNA
The double stranded DNA of interest separated from
the sample
Size range 100-35000 bp length

7. PRIMERS
2 sets of primers
Generally 20-30
nucleotides long
Synthetically produced
complimentary to the
3’ ends of target DNA
not complimentary to
each other

8. Enzymes
Usually Taq DNA Polymerase or anyone of the natural
or Recombinant thermostable polymerases
Stable at T° up to 95° C
High processivity
Taq Pol has 5’-3’ exo only
Thermus aquaticus

9. Deoxynucleotides
Single units of the bases A, T, G, and C (dATP, dTTP, dGTP,
dCTP)
They provide the energy for polymerization and the building
blocks for DNA synthesis.

10. Buffers
Magnesium & Potassium to provide the optimal
conditions for DNA denaturation and renaturation.
It also important for polymerase activity, stability.

11. Cycles in PCR
On rising the temperature to about 95° C/1 min
The DNA gets denatured
During this, the double stranded DNA is denatured
to single strands
Due to breakage in weak hydrogen bonds.
1. Denaturation

13. 2. Renaturation
The reaction temperature is rapidly lowered to 54-60°C for
20-40 seconds.
This allows the primers to bind (anneal) to their
complementary sequence in the template DNA.
Renaturation or Annealing. Binding depends conc. of
primers.

14. 3. Synthesis
Also known at extension or elongation
This step usually occurs at 72-80°C (most commonly 72°C).
The polymerase enzyme sequentially adds bases to the 3′ each
primer, extending the DNA sequence in the 5′ to 3′ direction.
Under optimal conditions, DNA polymerase will add about
1,000 bp/minute.

16. Standard Thermocycle

17. Target Amplification

18. Real-time PCR
Quantitative real time PCR (Q-RT PCR)
Reverse Transcriptase PCR (RT-PCR)
Multiplex PCR
Nested PCR
Long-range PCR
Single-cell PCR
Fast-cycling PCR
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR
In situ PCR
Variable Number of Tandem
Repeats (VNTR) PCR
Asymmetric PCR
Repetitive sequence-based PCR
Overlap extension PCR
Assemble PCR
Inter sequence-specific PCR(ISSR)
Ligation-mediated PCR
Methylation –specifin PCR
Mini primer PCR
Solid phase PCR
Touch down PCR, etc.
Types of PCR

19. Applications
Medical
Genetic testing for presence of genetic disease mutations. Eg:
hemoglobinopathies, cystic fibrosis, other inborn errors of metabolism.
 Detection of disease causing genes in suspected parents who act as
carriers.
Helps to monitor the gene in gene therapy
Analyzing clinical specimens for the presence of infectious agents,
including HIV, hepatitis, malaria, tuberulosis, and now COVID 19 etc.
Forensic
Can be used as a tool in genetic fingerprinting. This technology can
identify any one person from millions of others in case of : crime science,
rule out suspects during police investigation, paternity testing even in
case of availbility of very small amount of specimens ( stains of blood,
semen, hair etc)

20. Research and Molecular Genetics
1. In genomic studies: PCR helps to compare the genomes of two
organisms and identify the difference between them.
2. In phylogenetic analysis. Minute quantities of DNA from any
source such a fossilized material, hair, bones, mummified tissues.
3. In study of gene expression analysis, PCR based mutagenesis
4. In Human genome project for aim to complete mapping and
understanding of all genes of human beings.

21. References
U. Satyanarayana (2018) Biotechnology, Book and Allied
publication, Kolkata
Laboratory Info: https://laboratoryinfo.com/polymerase-chain-
reaction-pcr/ Accessed on 05.07.2020

22. Thank you